User:Brian P. Josey/Notebook/2010/02/10

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Ferritin

The ferritin came in while I was out. It is a brown solution with a slight dark red tint. I drew some out of the main bottle with a pippettor, and placed it in a stock tube. For future reference it is labeled on top as "FeS" for ferritin (and iron) stock. On the side I wrote out "Ferritin stock." In this tube I put in 100 uL.

I then created a dilution of the ferritin at 1:10 in water, 5 uL of the stock ferritin and 45 uL of DI water. Marked as "1:10 Fe" on top, and "1:10 Fe in H2O- 50uL" on the side. It is a lighter color than the stock, more of an amber than a red or a brown. Some of the ferritin did not go into solution right away, leaving a dark spot at the bottom of the tube. This was a very small portion, but it gave me a chance to try the magnet. I held up one of the small neodymium magnets to the side of the tube, and the dark spot was attracted to the magnet, in exactly the same way the beads were last Friday.

After mixing the tube together, I put the tube in an upside-down tube holder, and balanced that so the tube was placed between the tip of the nail and the neodymium magnet on the yoke. I had to put the rack upside-down so that I could see it. I am leaving it for one hour, 12:30-1:30, to see what happens. This should give the tube plenty of time to go through the magnetization process. What I hope happens is that either the tube becomes more clear, or the areas around both the tip and magnet become darker than the rest of the tube. Both of these would prove just how magnetic the ferritin is. If the tube remains uniform in color and darkness, then I will have to come back and rethink what I am doing.

Results

I let the tube sit for an hour and a half, half an hour longer that I originally planned. Unfortunately, there really wasn't any change in color, or development of protein clusters. From the best that I can tell, the only thing that happened, was the tube sat out on the lab bench for ninety minutes. To be perfectly honest, I'm not surprised. The set up that I had was very basic, and simple. It would have been cool to have a success like that right of the bat, but going into it, I knew that it would be unlikely. I am going to try to come up with some new ideas, and see where they can take me. I would like to see if I can visualize the proteins on the microscope. I doubt that I will, the proteins are very small, but getting some more hands-on with the microscope will help me out a lot down the road.

Microscope

I created a flow cell of my 1:10 dilution of the ferritin. Because the proteins are so small, on the order of 12 nm, I will not see the proteins individually. However, the main idea is see if I can see them as a whole. So I will look at the field of view, and then introduce a magnet. Hopefully, areas around the magnet will become darker, indicating heavy presence of the protein.

Results

Like I suspected, I was unable to see individual proteins. Considering their size, this isn't surprising in the least. We tried placing the magnet on the slide and seeing if there was any change in the field of view, and there wasn't. Andy then did a simplified version of dark field, by placing an opaque disk on the condenser. This darkened the field of view, but after placing the magnet on the slide, I didn't notice any changes. It looks like I am going to have to come up with another way to visualize the ferritin.

SJK 22:23, 10 February 2010 (EST)
22:23, 10 February 2010 (EST)Good experiments, seems like negative results so far.  I wouldn't write off any of them quite yet, and it's good to get your feet wet in each of them.  Some tweaks may help any of them.  I think doing some calculations would be good to get a foothold.  I've done all of these in the past, and there's a variety of them that would be good to write up here.  For example, one calculation is: given a force of __ pN, what "characteristic thickness" would you expect (accounting for thermal energy).  This is a relatively simple and useful calc to do.  We can also calculate forces, assuming certain gradients & fields, etc.  Actually those get more complicated and there's all kinds of very useful things you can learn--some labview and finite element modeling things.  All very cool, but some tricky things too!  We'll talk more / mindmap would help too!
22:23, 10 February 2010 (EST)
Good experiments, seems like negative results so far. I wouldn't write off any of them quite yet, and it's good to get your feet wet in each of them. Some tweaks may help any of them. I think doing some calculations would be good to get a foothold. I've done all of these in the past, and there's a variety of them that would be good to write up here. For example, one calculation is: given a force of __ pN, what "characteristic thickness" would you expect (accounting for thermal energy). This is a relatively simple and useful calc to do. We can also calculate forces, assuming certain gradients & fields, etc. Actually those get more complicated and there's all kinds of very useful things you can learn--some labview and finite element modeling things. All very cool, but some tricky things too! We'll talk more / mindmap would help too!
SJK 12:56, 11 February 2010 (EST)
12:56, 11 February 2010 (EST)To elaborate on why I said not to write them off: (a) for the in-tube experiment.  It's possible that there is some accumulation very near the side of the tube that we can't see.  Or it's possible the tube wall is too thick (try thin-walled tube).  For the actual experiments we want to do (in-cell), we can get much closer: this means higher field & higher gradient & thus higher force.  So, any accumulation you can see is a good sign.  We can get within 10's of microns of the proteins for the in-cell experiments.  And microfabricated magnets could perhaps do even better.
12:56, 11 February 2010 (EST)
To elaborate on why I said not to write them off: (a) for the in-tube experiment. It's possible that there is some accumulation very near the side of the tube that we can't see. Or it's possible the tube wall is too thick (try thin-walled tube). For the actual experiments we want to do (in-cell), we can get much closer: this means higher field & higher gradient & thus higher force. So, any accumulation you can see is a good sign. We can get within 10's of microns of the proteins for the in-cell experiments. And microfabricated magnets could perhaps do even better.


Using the Microscope

While I was using the microscope, Andy was able to help me expand upon what I learned on Friday. In the interest of having all my notes in one place, and having a reference for the future, here is a simple outline of everything we covered.

Setting Up the Microscope

Here are the steps to setting up the microscope:

  • Remove all of the covers
  • Put the objective in
  • Put a drop on the objective lens, enough to cover it
  • Replace the slide holder
  • Put the slide in the holder, coverslip down
  • Lift the objective up to the slide, and spread the oil around the area you are looking at
  • Turn on the light, focus and use the microscope

Köhler Illumination

I also went over how to set up Köhler illumination.

  • Find something to focus on
  • Push in the metal bar to get an extra lens
  • Move the condenser up and down until you see the edge
  • Adjust the condenser, using the side knobs until it is centered, and the aperture just traces out the edge of the field of view
  • Open the aperture so that it is just outside the field of view
  • Pull out the metal bar

Cleaning up and Closing Shop

Cleaning up the microscope after using it is essentially setting it up in reverse.

  • Turn off the lamp
  • Remove objective and clean with a kimwipe and isopropanol
  • Cap and store objective
  • Screw in little cap over objective
  • Replace all caps and covers
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